cgmp" of Genomic Microbial Identification

Transcription

1 cgmp" of Genomic Microbial Identification Presented by Wayne McKenzie & Tara Cassidy, August 2018 Slide 1 PharmOut 2018

2 Microgenetix Expertise in Microbial Genomics Recognized Market Leader and expert in Microbial Genomics / Identification Proven delivery in leading Pharma / Food / Medical Device / Consultant Laboratory market segments Reference laboratory for major Melbourne Hospital and leading consultant laboratories Strong regulatory experience cgmp / HACCP Expert staff - Microbiologists & Biotechnologists cgmp Compliant Facilities & Equipment Multiple Platform approach for Microbial Identification Validated and qualified systems 4500 sequencing runs 250,000 identifications in the last 10 years Early technology adopter first company in Australia to commercially sequence microorganisms Slide 2 PharmOut Microgenetix 2018

3 p U Dedicated Microbial Identification Laboratory MALDI- TOF Sample Preparation Sample Receipt Class II Safety Cabinet Incubator Incubator Staging Class II Safety Cabinet DNA Extraction GENOMICS GENERAL LABORATORY AREA Strain Typing Class II Safety Cabinet Real-time PCR PCR Preparation Class II Safety Cabinet Class II Safety Cabinet cgmp Compliant HEPA Filtered and segregated work zones PC2 PATHOGEN REFERENCE LABORATORY 0 Pa > 0 Pa -15 Pa Double barrier work zones with Incubator GENOMICS SEQUENCING ROOM GENOMICS PCR ROOM dedicated equipment LABORATORY MANAGER OFFICE 3130xl Genetic Analyzer Thermal Cycler Thermal Cycler Flush clean finish -80 C Freezer STORE ROOM RECEPTION Class II Safety Cabinet 3130xl Genetic Analyzer Class II Safety Cabinet Thermal Cycler Thermal Cycler Manometer Return Air Grille Ceiling HEPA Filter Validated equipment and procedures Proven Capacity and Redundancy Laboratory Bench Fully documented and implemented Quality System Dedicated and Controlled environment Microgenetix 2018 Slide 3 PharmOut 2018

4 Microbial Genomics at MGX % speciation rate 1800 bacterial species Library 500bp 16S sequence Limited identification platforms Limited knowledge of microbial diversity 26 region sequencing for Fungi 4 day result turnaround 2018 Over 250,000 isolates identified Close to 100% speciation rate 6000 MGX bacterial library 800bp 16S sequence Multiple identification platforms 26S/ ITS region sequencing for Fungi Next day result turnaround same day Limited client isolate history 2 MASM Microbiologist Significant client isolate history 7 MASM Microbiologists Microgenetix 2018 Slide 4 PharmOut 2018

5 Multiplatform Approach Sanger Sequencing - (2x 3130xl Genetic Analysers) MicroSEQ (10 years) MGX Library MGX 16S (5 years) MALDI-TOF Proteolytic Identification Species Identification Phylotyping Real Time PCR Methods (Specific Target) Probiotics Next Generation Sequencing Validated DNA Extraction 16S Diversity profiling (NGS) Whole Genome Sequencing REP PCR Strain Typing State of art Microbial Identification Slide 5 Microgenetix PharmOut

6 Gold Standard 16S Sequencing MicroSEQ - Validated System Workflow Starting material: sample from colony or pure culture DNA isolation with PrepMan Ultra kit PCR with MicroSeq PCR kit (1 reaction / sample) Purification of PCR product Sequencing reaction with MicroSeq sequencing kit (2 reactions / sample: forward and reverse) Purification of sequencing product Capillary electrophoresis on Genetic Analyzer Data analysis in MicroSeq ID software Library Search Report (Hit List) Slide 6 Microgenetix PharmOut

7 MGX 16S Sequencing Optimisation MicroSEQ delivers CFR part 11 compliance via Test Kit Reagents, Validated Equipment, Curated Library and Software. Following 10+ years and 100,000 s of identifications, MGX have optimised sequencing reflecting world best practice approaches including: MGX Bacterial Library Validated library - (6000+ entries with over MGX custom entries) provides close to a 100% next day speciation result History - MGX library accessions added from Australian isolates on an ongoing basis Tracking - Great comparison tool - we keep every sequence! High level of accuracy and reproducibility Improved species confidence influencing 41% of MicroSEQ species results eg. Microgenetix 2018 Slide 7 PharmOut 2018

8 Optimisation of Sanger Sequencing MGX 16S & ITS Generic Sequencing reagents Cost Effective - Non CFR compliant but uses the same validated equipment, systems, staff and software. MGX 16S Increased Accuracy via Longer Reads MGX QC Sanger sequencing data shows reliable long read lengths are achievable (up to 900 base pairs) A longer read inclusive of the MicroSEQ 500 (v1-v3) read achieves an additional base pair reads targeting more (v1,v2,v3 & v4) 16S gene regions providing: = Higher resolution = More informative than MicroSEQ sequence reads alone Longer reads overcome polymorphisms & short consensus issues (frequent with some genera eg. Bacillus sp.) Better exploits the powerful MGX 16S Full gene custom library Slide 8 Microgenetix PharmOut

9 MGX 16S 800 Validation Experience MGX Validation Results Highly accurate (100%) Highly reproducible (100%) Satisfies USP 1113 monograph requirement for verification of microbial identification methods Accuracy - Equivalent to MicroSEQ: over 95% of isolates (n>50) in the validation study obtaining the same top match best hit or an improved identification basis (>13%) Recommendations Result - high speciation outcome with next day result guarantee Improved identification basis for many genera, eg. many Bacillus species, and is expected to provide improved identification basis for microorganisms with polymorphisms in the 16s rdna gene Cost - Equivalent to true cost of MALDI TAT - fast turn around (next day) Existing MicroSEQ User Acceptance Criteria is appropriate Ongoing approach to library development Microgenetix 2018 Slide 9 PharmOut 2018

10 MALDI-TOF Proteolytic Identification (3 years) MALDI-TOF advantages seen for our clients: Bruker MALDI-TOF is CFR Compliant (cgmp) Rapid Same Day identification service Low sample presentation cost with Direct Method Phylotyping capability for some organisms MALDI-TOF challenges: Performance Issues - Colony age, Sample re-streaking time delays up to 5 days with fall through (Up to 15%) requiring sequencing Sample Preparation / extraction methods - can improve reproducibility Score Value Poor consensus re acceptance criteria (OEM / IVD) Poor demarcation / reproducibility with some genera Lower speciation confidence MSP Library Updates / Suitability Necessary with relevance to client environs Service Issues - laser tuning result & costs impacts MALDI-TOF More suitable for less critical environments / screening Microgenetix 2018 Slide 10 PharmOut 2018

11 MALDI-TOF Strain typing (P.acnes) Flex Analysis Offline Software Slide 11 Microgenetix PharmOut 2018

12 Nucleic Acid Based Techniques Increasingly pharmacopoeia are providing for NAD / Genotypic Microbial Identification Methods USP 113 describes methods to validate and verify nucleic acid test assays DNA genotyping Microbial Identification Traditional PCR Active Identification - confirmation of probiotic / vaccine strains Real-Time PCR targeted detection of biological contaminants e.g. Mycoplasma Microgenetix 2018 Slide 12 PharmOut 2018

13 USP dietary supplement and FCC monographs for probiotic strains USP published 8 monographs in 2015 in the Food Chemical Codex (FCC) which provides an active specificity test challenging the suitability of many probiotics Lactobacillus acidophilus LA-14 & NCFM Lactobacillus rhamnosus HN001 Lactobacillus paracasei Lpc-37 Bifidobacterium animalis ssp. lactis (HN019, Bi-07, Bl-04) Monographs contain (At strain level): Description Identification Assay Specific Tests Labelling requirement Bacillus coagulans GBI-30,6086 Slide 13 Microgenetix PharmOut

14 Nucleic Acid-Based Identification of Probiotic Strains Identification Process 1. Targeted primer sets 2. PCR Amplification 3. DNA Electrophoresis 4. DNA Sequencing (SNP Analysis) Primer set 1 Primer set 2 Acceptance Criteria for B.lactis HN019 PCR Preparation for primer set 1 gives an amplification product of 351 base pairs with a SNP identified as CTTCAGATTTTAGGC PCR Preparation for primer set 2 gives an amplification product of 531 base pairs with a SNP identified as GCCCGCTCAAACGAA Microgenetix 2018 Slide 14 PharmOut 2018

15 NGS Next Generation Sequencing Applications Environmental monitoring (best practice) Gut Microbiome (Humans & Animals) Probiotic organism identification / evaluation Fermented Foods & Beverages (Control / Drift) Whole genome (Trace - Highly Specific) Key Measurements DAS Microbial Diversity, Abundance and Significance Slide 15 Microgenetix PharmOut

16 Microbial Diversity ATCC Mock Community Results MGX deliver world class best practice Microbial Diversity Slide 16 Microgenetix PharmOut 2018

17 NGS Microbial Diversity provides a best practice (Illumina - 2x300bp reads) approach to environmental monitoring - Surface Swab Results 16S Bacterial Diversity ex Swab ITS Fungal Diversity ex Swab Sample no.: Sample no.: Slide 17 Microgenetix PharmOut

18 Gut Microbiome Microbial Diversity Believed to have huge impacts on health and wellbeing being implicated in many diseases and disorders. Human Gut Phylum Level Comparison Human Gut Genus Level Comparison 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Actinobacteria Bacteroidetes Chloroflexi Cyanobacteria Euryarchaeota Firmicutes Nitrospirae Proteobacteria Tenericutes Unclassified at Phylum level Verrucomicrobia 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Alkaliphilus Bacteroides Bifidobacterium Blautia Catenibacterium Collinsella Coprococcus Faecalibacterium Methanobrevibacter Oscillospira Phascolarctobacterium Prevotella Ruminococcus Succinivibrio Unclassified at Genus level Slide 18 Microgenetix PharmOut

19 Probiotics / fermented foods Microbial diverse samples - Can be accurately tested for the first time! Slide 19 Microgenetix PharmOut

20 NGS Challenges met by cgmp approach Sampling DNA Extraction Primer Selection Sequencing Approach Bioinformatics selection Microgenetix 2018 Slide 20 PharmOut 2018

21 NGS & Bionformatics MGX Advanced Pipeline / Significant organisms C.difficle ABUNDANCE (%) C.botulinum ABUNDANCE (%) TEST SAMPLE MGX MSR/BaseSpace QIIME MGX MSR/BaseSpace QIIME Slide 21 Microgenetix PharmOut

22 Whole Genome Sequencing Tracing Whole genome sequencing reveals the complete DNA make-up of an organism, enabling us to better understand variations both within and between species. Provides a high-resolution, base-by-base view of the genome Captures both large and small variants that might otherwise be missed Identifies potential causative variants for further follow-on studies of gene expression and regulation mechanisms Delivers large volumes of data in a short amount of time to support assembly of novel genomes Microgenetix 2018 Slide 22 PharmOut 2018

23 Thankyou Wayne McKenzie / Tara Cassidy Microgenetix wayne@microgenetix.com.au Slide 23 PharmOut 2018